Treponema pallidum subsp. pallidum and related organisms are helically- shaped bacteria which cause venereal syphilis, endemic syphilis, yaws and pinta in humans. Although the incidence of sexually transmitted diseases would be expected to decrease with changing sexual behavior as a result of the AIDS epidemic, the number of reported primary and secondary syphilis cases in the United States increased 30% in 1987, due primarily to apparent epidemics in New York, Florida, and California. Further understanding of T. pallidum is needed to aid in the prevention, diagnosis, and treatment of syphilis. In the proposed research, we will continue studies on the physiology, structure, and antimicrobial susceptibility of T. pallidum. The principal focus will be efforts to continuously culture T pallidum in vitro, using as a starting point a tissue culture system which supports 20- to 100-fold multiplication of the organism over a 12 to 15 day period of incubation. Mammalian cells, in particular Sf1Ep cottontail rabbit epithelial cells, are required for the in vitro multiplication of T. pallidum in this system, but recent studies indicate that rapid growth or high metabolic rates of these cells inhibit treponemal multiplication. Furthermore, removal of amino acids and vitamins from the culture medium enhances the multiplication of T. pallidum, apparently by inhibiting the growth and metabolism of the tissue culture cells. In the coming grant period, conditions inhibitory to mammalian cell metabolism, including irradiation, addition of cycloheximide, and use of nutrient-deficient media, will be tested for their ability to enhance the multiplication of T. pallidum If conditions are identified which significantly increase and prolong treponemal growth, they will be combined with subculture or medium replacement techniques in an attempt to produce continuous growth. Through the deletion of medium components, dialysis of serum, and removal of tissue extract, it should be possible to define the nutritional requirements of T pallidum. In addition, the biosynthetic capacity of T. pallidum will be assessed by measuring the incorporation of radiolabelled D-glucose, fatty acids, and inorganic salts into proteins, nucleic acids, and lipids. To assess the antimicrobial susceptibility pattern of T. pallidum, the in vitro culture system will be utilized to determine the minimal inhibitory and bactericidal concentrations of a variety antimicrobial agents against the Nichols strain and other pathogenic strains. In our continuing studies of the major structural elements of T. pallidum, we will attempt to purify and characterize the outer membrane, cytoplasmic membrane, and cytoplasmic fibrils of pallidum using isoelectric focusing, hydrophobic interaction chromatography, sucrose gradient sedimentation, and other methods. Immunoelectron microscopy of ultrathin sections will be used to localize both internal and external proteins in the overall structural studies will be integrated with recent immunologic and recombinant DNA studies to improve our understanding of the host-parasite interactions of this unusual organism.